Figure 1.

IRSp53 slows down barbed end growth: an effect relieved by CDC42. (A) IRSp53 and VASP form large clusters in solution. Intensity weighted diameter distributions for IRSp53 and VASP showed single species for either IRSp53 (red lines) or VASP WT and mutant proteins alone at concentrations of up to 30 μM (green lines). Aggregates formed in mixtures of 10 μM IRSp53 and 10 μM VASP WT or VASPΔ(GP5)₃, whereas no clusters were observed with VASP-ΔPRD (blue lines). (B) VASP increases the association rate constant of profilin-actin to barbed ends. Barbed end growth was measured in bulk pyrenyl-actin polymerization assays using 1.25 μM MgATP-G-actin (5% pyrenyl-labelled), 5 μM profilin, and VASP (V) at 0 (blue lines), 64 nM (red lines), and 169 nM (green lines), in the absence (dotted lines) or in the presence (continuous lines) of 0.16 nM spectrin-actin seeds. Inset shows cumulated data from additional assays performed with different VASP concentrations. (C) IRSp53 slows down actin polymerization by weakly capping barbed ends. Kinetics of actin polymerization induced by spectrin-actin seeds, measured as described in (A), in the presence of the indicated concentrations of IRSp53. (D) IRSp53 and VASP inhibit barbed end depolymerization. Depolymerization of actin filaments (2.5 μM F-actin, 50% pyrenyl-labelled) was induced by 50-fold dilution into polymerization buffer in the presence of 0.29 μM IRSp53 (red curve) or 0.34 μM VASP (green curve). (E) Summary of barbed end growth inhibition (Cap) by the indicated IRSp53 constructs and mutants (see also Supplementary Figure S2). (F) Active CDC42 relieves IRSp53 capping activity. Kinetics of actin polymerization induced by spectrin-actin seeds was measured as described in (A) in the presence of 0.58 μM IRSp53, alone or with 10 μM CDC42-GTP or CDC42-GDP. Control (CTR) sample contained no IRSp53. (G) Kinetics of actin polymerization induced by spectrin-actin seeds was measured as described in (A) in the presence of the indicated concentrations of IRSp53 or its truncated mutant 1–375. Right graph represents a magnification of the initial phase of elongation by seeds (inset in the left graph).